Process for extracting procainamide from blood

ABSTRACT

Procainamide may be extracted from blood by a process which comprises first mixing the blood with an alcoholic base, followed by the addition of a solvent, addition of a water-soluble colloid, and separation of the solvent which contains the procainamide.

United States Patent [191 Klinger [4 1 Feb. 25, 1975 PROCESS FOR EX TRACTING PROCAINAMIDE FROM BLOOD [75] Inventor: Harry Klinger, Summit, NJ.

[73] Assignee: E. R. Squibb & Sons, Inc.,

Princeton, NJ.

[22] Filed: Aug. 13, 1973 [21] Appl. No.: 387,546

[52] U.S. Cl. 23/230 B, 260/684, 260/705 [51] Int. CL. B01d 11/00, G0ln 31/02, GOln 33/16 [58] Field of Search 23/230 B; 260/112 B, 122,

[56] References Cited UNITED STATES PATENTS 3,681,283 8/1972 Yueh 210/54 X 3,706,660 12/1972 Hagan et al 210/54 X OTHER PUBLICATIONS Clinical Chemistry, Vol. 18, p. 643, Rapid Gas Chromatographic Measurement of Plasma Procainamide Concentration, Atkinson, Jr. et al.

Journal of the American Chemical Society, Vol. 66, p. 692-697, 1944, The Precipitation of Proteins by Synthetic Detergents, Putnam et al.

Primary Examiner-Morris O. Wolk Assistant Examiner-Michael S. Marcus Attorney, Agent, or FirmLawrence S. Levinson; Merle J. Smith; Donald J. Barrack [57] ABSTRACT 11 Claims, No Drawings BACKGROUND OF THE INVENTION Procainamide is an antiarrhythmic agent, the effects of which are particularly beneficial in treating ventricular arrhythmias. Ventricular extrasystoles and ventricular tachycardia may be controlled within an hour after oral or intramuscular administration or within a few minutes after intravenous infusion of the drug. By judicious administration of procainamide, digitalis-induced ventricular extrasystoles and tachycardia may at times be controlled. Procainamide may also be of value in the control of an auricular arrhythmia.

It is preferred that procainamide be administered orally. However, parenteral administration (e.g., intramuscular) may be necessary. While suggested dosages are available to the physician, each patient must of course be treated as a separate case. It is generally felt that blood levels of procainamide ranging from about 4 parts per million (hereinafter ppm.) to about 7 ppm. are optimal.

At present there are methods known for quantitatively measuring procainamide blood levels. One such method is the modified Bratton and Marshall procedure; see Journal of Pharmacology and Experimental Therapeutics 102, -15 (1951). Other methods for the quantitative determination of procainamide utilize gas chromatography (see Clinical Chemistry 18, 643 (1972)) and fluorimetry (see Journal of the American Medical Associatin 215 (9), 1454 (1971)). As now run, the above procedures require relatively large amounts of blood (i.e., about 3 to 10 milliliters) and a centrifuge for separation purposes.

It is the purpose of this invention to enable doctors (or nurses or technicians) to make a quantitative determination of procainamide in blood using a few drops (or even as little as a'fraction of a drop) of blood. A bedside test for determining procainamide blood levels is particularly desirable because of the urgency of treatment for patients suffering from a cardiac arrhythmia.

SUMMARY OF THE INVENTION The problem to which this invention is directed is the removal of procainamide from small quantities of blood by a procedure which can be carried out in an efficient manner without the use of a centrifuge or similar separation equipment. Once the procainamide is removed from the blood, a quantitative analysis may be carried out.

The process of this invention is a process for extracting procainamide from blood and may be used on as little as a fraction of a drop of blood. The process comprises basically four steps that may be summarized as follows:

i. mixing a blood sample with alcoholic alkali;

ii. adding a solvent to the mixture and stirring;

iii. adding a water-soluble colloid to the mixture and stirring; and

iv. separating the solvent from the blood residue. (The expression blood residue" is used here, and throughout the specification, to describe the residue remaining after the solvent containing procainamide is separated from the extraction mixture.) To assure that all of the procainamide has been removed from the blood sample, the blood residue may be re-extracted one or more times with additional solvent.

DETAILED DESCRIPTION OF THE INVENTION The size of the blood sample to be analyzed for procainamide is not critical, although it is important that the exact amount of the sample be known. It is an important advantage of this invention that the extraction process may be run on very small amounts of blood (as little as about /2 drop). Generally, the blood sample will be about 25 to microliters (about to 3 drops).

The blood sample is first mixed with an alcoholic alkali. The amount of alcoholic alkali used is not critical, but generally the amount of alkali used will be approximately equal in volume to the blood sample. The purpose of the alcoholic alkali is to convert the procainamide salt (procainamide is in the blood in the form of a salt) to the free base. The base used should be a strong one; sodium hydroxide, potassiium hydroxide, and tetramethylammonium hydroxide are preferred, with sodium hydroxide and potassium hydroxide being particularly preferred.

After stirring the alcoholic alkali with the blood sample, a solvent is added to the mixture. Any solvent may be used as long as: (i) it is water-immiscible and (ii) it will dissolve procainamide base. Exemplary of the solvents which may be used in the process of this invention are aliphatic hydrocarbons (e.g., pentane and hexane), halogenated hydrocarbons (e.g., chloroform and methylene chloride), petroleum ether, and aromatic hydrocarbons (e.g., benzene and toluene). The halogenated hydrocarbons are preferred solvents, chloroform and methylene chloride being particularly preferred. As will be appreciated by one skilled in the art, the volume ratio of solvent to blood sample is not critical to this invention. Generally, however, a volume ratio of solvent to blood will be between about 6:1 and about 10:1. After adding the solvent to the blood sample/alcoholic alkali mixture, the entire mixture is again stirred.

The above mixing facilitates passage of procainamide from the alkalinized blood into the solvent. However, at this stage of the extraction process the blood has a tendency to float on the solvent surface and small fibrous particles are sometimes distributed throughout the solvent layer. These problems make a clean separation of the solvent layer from the blood residue difficult.

To facilitate a clean separation of the solvent layer from the blood residue a water-soluble colloid is added to the vessel (in which the extraction is carried out) containing the blood sample, alcoholic alkali, and solvent. The water-soluble colloid absorbs the excess water in the blood and forms a small mass containing the blood solids. This mass will rest on the bottom of the vessel allowing the solvent layer (containing the procainamide) to be poured off.

The expression water-soluble colloid as used herein is a term of art. The materials are not watersoluble in the same way as, for example, salt or sugar, but rather they swell when they come into contact with water. The water-soluble colloid that may be used in the process ofthis invention may be chosen from synthetic materials or from natural gums. Exemplary of the synthetic materials contemplated for use in this invention are sodium carboxymethylcellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and polyvinyl alcohol. Exemplary of the natural gums are locust bean, guar, acacia, karaya, ghatti, agar, algin, pectin, xanthan, and psyllium seed. The synthetic water-soluble colloids are preferred since they are usually obtainable in purer form than the natural gums. The impurities often present in natural gums prevent them from swelling as cleanly in water as the synthetic colloids.

Mixing of the water-soluble colloid with the blood sample, alcoholic alkali, and solvent proceeds until the colloid has formed a mass. The solvent containing the procainamide is then poured off.

To be certain that all of the procainamide has been removed from the blood sample additional solvent may be mixed with the blood residue, and then poured off and combined with the prior extract. This procedure may be repeated more than once.

Essentially all of the procainamide originally in the blood sample is extracted by the procedure of this invention into the solvent. The amount of procainamide in solution may be determined using methods known to those skilled in the art.

The following examples are presented to further illustrate this invention.

Example 1 Using a blood lancet, the fingertip skin is pierced and the finger is squeezed until a drop of blood accumlates. 50 ul of blood is drawn from the drop into a disposable pipet and is blown into a 1 ml. beaker.

A drop of a 0.2 percent solution of potassium hydroxide in methanol is added to the beaker, and the mixture is stirred with a glass stirring rod. 0.3 ml. of methylene chloride is added to the beaker and the mixture is stirred well. mg. of soidum carboxymethylcellulose (Cellulose Gum 7H, a-product of Hercules, Inc.) is added to the breaker and the mixture is again stirred. (The carboxymethylcellulose forms a gelatinous blob). Stirring is continued until no floating particles are observed, and the clear methylene chloride is then poured off into a clean l ml. beaker.

A second 0.3 ml. portion of methylene chloride is added to the first beaker, stirred, and the methylene chloride poured off into the second beaker to mix with the first portion. (At this stage, essentially all of the procainamide from the 50 ul. of blood is dissolved in the methylene chloride.)

Example 2 Using a blood lancet, the fingertip skin is pierced and the finger is squeezed until a drop of blood accumulates. 50 ul. of blood is drawn from the drop into a disposable pipet and is blown into a 1 ml. beaker.

A drop of a 0.2 percent solution of potassium hydroxide in methanol is added to the beaker, and the mixture is stirred with a glass stirring rod. 0.3 ml. of chloroform is added to the beaker and the mixture is stirred well. 5 mg. of sodim carboxymethylcellulose (Cellulose Gum 7H, a product of Hercules, Inc.) is added to the beaker and the mixture is again stirred. (The carboxymethylcellulose forms a gelatinous blob). Stirring is continued until no floating particles are observed, and the clear chloroform is then poured offinto a clean 1 ml. beaker.

A second 0.3 ml. portion of chloroform is added to the first beaker, stirred, and the methylene chloride poured off into the second beaker to mix with the first portion. (At this stage, essentially all of the procainamide from the 50 ul. of blood is dissolved in the chloroform.

Example 3 Using a blood lancet, the fingertip skin is pierced and the finger is squeezed until a drop of blood accumulates. 50 ul. of blood is drawn from the drop into a disposable pipet and is blown into a 1 ml. beaker.

A drop of a 0.2 percent solution of sodium hydroxide in methanol is added to the beaker, and the mixture is stirred with a glass stirring rod. 0.3 ml. of methylene chloride is added to the beaker and the mixture is stirred well. 5 mg. of sodium carboxymethylcellulose (Cellulose Gum 7H, a product of Hercules, Inc.) is added to the beaker and the mixture is again stirred. (The carboxymethylcellulose forms a gelatinous blob). Stirring is continued until no floating particles are observed, and the clear methylene chloride is then poured off into a clean 1 ml. beaker.

' A second 0.3 ml. portion of methylene chloride is added to the first beaker, stirred, and the methylene chloride poured off into the second beaker to mix with the first portion. (At this stage, essentially all of the procainamide from the 50 ul. of blood is dissolved in the methylene chloride.)

Example 4 Using a blood lancet, the fingertip skin is pierced and the finger is squeezed until a drop of blood accumulates. 50 ul. of blood is drawn from the drop into a disposable pipet and is blown into a 1 ml. beaker.

A drop of a 0.2 percent solution of potassium hydroxide in methanol is added to the beaker, and the mixture is stirred with a'glass stirring rod. 0.3 ml. of methylene chloride is added to the beaker and the mixture is stirred well. 5 mg. of methyl cellulose is added to the beaker and the mixture is again stirred. (The methyl cellulose forms a gelatinous blob). Stirring is continued until no floating particles are observed, and the clear methylene chloride is then poured off into a clean 1 ml. beaker.

A second 0.3 ml. -portion of methylene chloride is added to the first beaker, stirred, and the methylene chloride poured off into the second beaker to mix with the first portion. (At this stage, essentially all of the procainamide from the 50 ul. of blood is dissolved in the methylene chloride.)

Example 5 Using a blood lancet, the fingertip skin is pierced and the finger is squeezed until a drop of blood accumulates. 50 ul. of blood is drawn from the drop into a disposable pipet and is blown into a 1 ml. beaker.

A drop of 0.2 percent solution of sodium hydroxide in methanol is added to the beaker, and the mixture is stirred with a glass stirring rod. 0.3 ml. of chloroform is added to the beakerand the mixture is stirred well. 5 mg. of pectin is added to the beaker and the mixture is again stirred. (The pectin forms a gelatinous blob). Stirring is continued until no floating particles are observed, and the clear chloroform is then poured off into a clean 1 ml. beaker.

A second 0.3 ml. portion of chloroform is added to the first beaker, stirred, and the chloroform poured off into the second beaker to mix with the first portion. (At this stage, essentially all of the procainamide from the 50 ul. of blood is dissolved in the chloroform.)

The following examples illustrate how the procainamide extracted from a drop of blood can be quantitatively measured.

Example 6 The 1 ml. beaker of Example 1 containing the methylene chloride solution of procainamide is placed on a heating unit and evaporated to dryness.

A drop of 0.1 N hydrochloric acid is added to the residue and the beaker is shaken to effect solution of the residue.

A drop of 0.2 percent sodium nitrite solution is added to the beaker. The beaker is shaken and then allowed to stand for 1 minute.

A drop of 10% ammonium sulfamate solution is added to the beaker. The beaker is shaken and then allowed to stand for 1 minute.

A drop of N-(1-naphthyl)ethylenediamine hydrochloride is added to the beaker.

After several minutes, the color of the mixture in the beaker is compared with a set of standards prepared from 50 ul. portions of procainamide solutions containing 2,4,8, and 12 parts per million of procainamide.

Example 7 A paper disc (18 mm. diameter) is cut from filter paper (Whatman No. 120) and fitted tightly into a 22 mm. polyethylene snap cap to, act as a receptacle. 4 Drops of a 2.5 percent aqueous solution of sodium bisulfate are added to the disc, which is then allowed to dry for 24 hours.

A drop of a 0.02 percent solution of dimethylaminocinnamaldehyde in methanol is added to a methylene chloride solution of procainamide in a 1 ml. beaker (prepared as in Example 1). The resultant solution is poured onto the treated paper disc in the receptacle and it is evaporated using heat provided by an electric lamp.

When the disc is completely dry, its color is compared with a set of standards prepared from 50 ul. portions of procainamide solutions containing 2,4,8, and 12. part per million of procainamide and treated as above.

I claim:

1. A process for extracting procainamide from blood that comprises:

(i) mixing a blood sample with alcoholic alkali;

(ii) adding to the mixture of (i) a water-immiscible solvent which will dissolve the procainamide in the blood;

(iii) adding a water-soluble colloid to the mixture of blood sample, alcoholic alkali, and waterimmiscible solvent; and

(iv) separating the solvent from the blood residue.

2. A process in accordance with claim 1 wherein the water-soluble colloid is a natural gum.

3. A process in accordance with claim 1 wherein the water-soluble colloid is selected from the group consisting of sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, and polyvinyl alcohol.

4. A process in accordance with claim 3 wherein the water-soluble colloid is sodium carboxymethylcellulose.

5. A process in accordance with claim I wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium hydroxide, alcoholic potassium hydroxide, and alcoholic tetramethylammonium hydroxide.

6. A process in accordance with claim 5 wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium hydroxide and alcoholic potassium hydroxide.

7. A process in accordance with claim 1 wherein the water-immiscible solvent is selected from the group consisting of aliphatic hydrocarbons, halogenated hydrocarbons, petroleum ether, and aromatic hydrocarbons.

8. A process in accordance with claim 7 wherein the water-immiscible solvent is a halogenated hydrocarbon.

9. A process in accordance with claim 8 wherein the water-immiscible solvent is selected from the group consisting of chloroform and methylene chloride.

10. A process in accordance with claim 1 which comprises re-extracting the blood residue with additional solvent.

1 l. A process in accordance with claim 1 wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium, alcoholic potassium and alcoholic tetramethylammonium hydroxide; the water-soluble colloid is selected from the group consisting of natural gum, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and polyvinyl alcohol; and the water-immiscible solvent is selected from the group consisting of aliphatic hydrocarbons, halogenated hydrocarbons, petroleum ether and aromatic hydrocarbons.

UNITED STATES PATENT OFFICE I 1 CERTIFICATE OF CORRECTION PATENT NO. 3,868,220 DATED February 25, 1975 INVENTOR(S) Harry Klingel It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

On abstract page, inventor's name should read--Klingel-.

Column 1, line 31, "Associatin" should rsad-Association-a Column 2, line 18, "potassiium" should readpotassium-.

Column 3, line 28, "accumlates" should read-accumulates.

Column 4, line 2, insert parentheses after the word "form."- to read--form.)-.

Column 6, line 41, "alcoholic sodium" should read-alcoholic sodium hydroxide--; "alcoholic potassium" should read alcoholic potassium hydroxide-.

Signed and sealed this 27th day of May 1975.

(SEAL) Attest:

c. MARSHALL DANN Commissioner of Patents and Trademarks RUTH C. MASON Attesting Officer 

1. A PROCESS FOR EXTRACTING PROCAINAMIDE FROM BLOOD THAT COMPRISES: (I) MIXING A BLOOD SAMPLE WITH ALCOHOLIC ALKALI; (II) ADDING TO THE MIXTURE OF (I) A WATER-IMMISCIBLE SOLVENT WHICH WILL DISSOLVE THE PROCAINAMIDE IN THE BLOOD; (III) ADDING A WATER-SOLUBLE COLLOID TO THE MIXTURE OF BLOOD SAMPLE, ALCOHOLIC ALKALI, AND WATER-IMMISCIBLE SOLVENT; AND (IV) SEPARATING THE SOLVENT FROM THE BLOOD RESIDUE.
 2. A process in accordance with claim 1 wherein the water-soluble colloid is a natural gum.
 3. A process in accordance with claim 1 wherein the water-soluble colloid is selected from the group consisting of sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, and polyvinyl alcohol.
 4. A process in accordance with claim 3 wherein the water-soluble colloid is sodium carboxymethylcellulose.
 5. A process in accordance with claim 1 wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium hydroxide, alcoholic potassium hydroxide, and alcoholic tetramethylammonium hydroxide.
 6. A process in accordance with claim 5 wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium hydroxide and alcoholic potassium hydroxide.
 7. A process in accordance with claim 1 wherein the water-immiscible solvent is selected from the group consisting of aliphatic hydrocarbons, halogenated hydrocarbons, petroleum ether, and aromatic hydrocarbons.
 8. A process in accordance with claim 7 wherein the water-immiscible solvent is a halogenated hydrocarbon.
 9. A process in accordance with claim 8 wherein the water-immiscible solvent is selected from the group consisting of chloroform and methylene chloride.
 10. A process in accordance with claim 1 which comprises re-extracting the blood residue with additional solvent.
 11. A process in accordance with claim 1 wherein the alcoholic alkali is selected from the group consisting of alcoholic sodium, alcoholic potassium and alcoholic tetramethylammonium hydroxide; the water-soluble colloid is selected from the group consisting of natural gum, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and polyvinyl alcohol; and the water-immiscible solvent is selected from the group consisting of aliphatic hydrocarbons, halogenated hydrocarbons, petroleum ether and aromatic hydrocarbons. 